CN105586303A - Korean pseudomonas and application thereof - Google Patents
Korean pseudomonas and application thereof Download PDFInfo
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Abstract
The invention provides a Korean pseudomonas and application thereof. The Korean pseudomonas is identified as Korean pseudomonas CJ-361, and the preservation number is CGMCC NO.12122. The Korean pseudomonas CJ-361 is obtained through separation from radix pseudostellariae rhizosphere soil by adopting a pseudomonas selectivity culture medium, an antagonistic experiment is performed on fusarium oxysporum and fusarium moniliforme separated to the radix pseudostellariae root lesion portion through an agar plate opposite culture method, and it is found that the Korean pseudomonas has the strong antagonism on the fusarium oxysporum and the fusarium moniliforme. Meanwhile, the invention provides the optimum culture medium for antagonistic bacterium growth, the optimum formula is 1/4LB+1/20MS (containing no ferric salt)+0.1 wt% brown sugar, and the fermented liquor has the obvious antagonistic effect. The antagonistic strain and the microbial agent thereof can effectively prevent and treat radix pseudostellariae root rot diseases, and the environment-friendly, ecological and safe biological prevention and control potential strain is provided for overcoming or relieving replantation diseases of radix pseudostellariae.
Description
Technical field
The present invention relates to a strain bacterial isolates, particularly strain Korea S pseudomonad and an application thereof, can be used for overcoming orAlleviate continuous cropping radix pseudostellariae soil-borne disease problem, belong to microorganism and biological prevention field.
Background technology
Radix pseudostellariae (Pseudostellariaheterophylla) is called virgin ginseng, caryophyllaceous ginseng, meter Can, for Caryophyllaceae for many yearsThe effects such as raw herbaceous plant, is used as medicine with root, has and replenishes qi to invigorate the spleen, promoting production of body fluid and nourishing the lung, its medication is with a long history, clinical efficacy is trueCut, determined and list " the Chinese medicine list that can be used for health food " in by the Ministry of Public Health at present. Radix pseudostellariae main product in Fujian, Guizhou,The ground such as Anhui, wherein long, best in quality with Zherong County, Fujian Province cultivation history, have " township of Chinese radix pseudostellariae " U.S.Reputation. There is serious Soil-sickness Problem in radix pseudostellariae, continuous cropping radix pseudostellariae plant strain growth is bad, underground part in cultivating and growing processCan not normally expand (as shown in Figure 1), after results, must after 2 ~ 4 years, can plant again. Soil-sickness Problem also causes a series ofThe bad problem in downstream, dwindles just year by year as radix pseudostellariae Genuine producing area and scale, even occurs moving outside producing region, and genuineness distortion etc. are existingResemble. Therefore, Soil-sickness Problem research is the ecological important content urgently to be resolved hurrily of current resources of medicinal plant, becomes both at home and abroadThe focus of colleague's research. Seminar's early-stage Study discovery, Fusarium oxysporum, fusarium moniliforme (as shown in Figure 2) are to cause crown princeTwo class important pathogen of ginseng continuous cropping obstacle, its content all increases and constantly increases with the radix pseudostellariae continuous cropping time limit. And utilize antagonismThe biological control that bacterium carries out plant disease is considered to a kind of science, reasonable, efficient, environmentally friendly measure. Pin of the present inventionTo radix pseudostellariae specialized form Fusarium oxysporum and fusarium moniliforme, separation screening is short of money to this two classes disease fungus of plant height effect antagonismAntibacterium, through being accredited as Korea S pseudomonad, called after PseudomonaskoreensisCJ-361.
Summary of the invention
The object of this invention is to provide strain Korea S pseudomonad and an application thereof, plant continuously for biological control radix pseudostellariaeCommon root rot problem in process, provides biocontrol bacterial strain for overcoming or alleviating radix pseudostellariae Soil-sickness Problem.
Technical scheme of the present invention is as follows:
Antagonistic bacterium provided by the present invention is Korea S pseudomonad (Pseudomonaskoreensis) CJ-361, this bacterial strainIn on January 28th, 2016 be preserved in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica inState's microorganism fungus kind preservation administration committee common micro-organisms center, deposit number is: CGMCCNO.12122.
Korea S of the present invention pseudomonad (PseudomonaskoreensisCJ-361) is to radix pseudostellariae specialized formPathogen-Fusarium oxysporum, fusarium moniliforme all have very strong antagonism.
The growth medium of described Korea S pseudomonad be 1/4LB culture medium+1/20 containing molysite MS culture medium+0.1wt.% brown sugar.
Described containing its formula of molysite MS culture medium is not: 1) a great number of elements: 1900mg/LKNO3、1650mg/LNH4NO3、370mg/LMgSO4·7H2O、170mg/LKH2PO4、440mg/LCaCl2·2H2O; 2) trace element: 22.3mg/LMnSO4·4H2O、8.6mg/LZnSO4·7H2O、6.2mg/LH3BO3、0.83mg/LKI、0.25mg/LNa2MoO4·7H2O、0.025mg/LCuSO4·5H2O、0.025mg/LCoCl、2.6mg/LH2O; 3) organic matter: 2.0Mg/L glycine, 0.5mg/L puridoxine hydrochloride, 0.1mg/L Tyiamine Hd element, 0.5mg/L nicotinic acid, 100mg/L fleshAcid; Described LB culture medium is: tryptone 10g/L, yeast extract 5g/L, NaCl10g/L.
Useful Antagonistic Fungi of the present invention has following advantage:
(1) Antagonistic Fungi-Korea S pseudomonad of the present invention (Pseudomonaskoreensis) CJ-361 is by greatlyThe screening operation of amount screens, and screens and obtains, to radix pseudostellariae specialized form Fusarium oxysporum from 1600 strain soil bacteriasAnd fusarium moniliforme all has very strong antagonistic effect (as shown in Figure 3).
(2) Antagonistic Fungi-Korea S pseudomonad of the present invention (Pseudomonaskoreensis) CJ-361 hasStronger cellulose degradation ability and phosphorus decomposing ability (as shown in Figure 4).
(3) Antagonistic Fungi-Korea S pseudomonad of the present invention (Pseudomonaskoreensis) CJ-361 sourceIn continuous cropping radix pseudostellariae rhizosphere soil, be not derived from other habitats, rhizosphere colonization effect is better and safe and reliable, to radix pseudostellariaeAnd rear other crops of stubble (as often planted crop in the radix pseudostellariae Genuine producing area such as paddy rice, soybean, corn, vegetables) are all infected energy without causing a diseasePower.
(4) training of Antagonistic Fungi-Korea S pseudomonad of the present invention (Pseudomonaskoreensis) CJ-361Foster temperature range is preferably 4 DEG C～45 DEG C, and pH scope is 5～9.5, and the temperature and the pH scope that are applicable to growth are wider, strong adaptability.
Brief description of the drawings
Fig. 1 is the main crop, continuous cropping radix pseudostellariae underground part piece root growth situation.
Fig. 2 is the bacterium colony shape of the pathogen (Fusarium oxysporum, fusarium moniliforme) that is separated to of radix pseudostellariae plant diseases rootState .A, B: represent respectively Fusarium oxysporum bacterium colony obverse and reverse; C, D: represent that respectively the front of fusarium moniliforme bacterium colony is with anti-Face.
Fig. 3 is that photograph is cultivated in the face-off of Korea S pseudomonad (Pseudomonaskoreensis) CJ-361 and disease fungusSheet. left side is dull and stereotyped is control group (only inoculating pathogen), and all kinds of disease funguses are inoculated, wherein A in dull and stereotyped center, right side: sharp spore sickleCutter bacterium (source radix pseudostellariae); B: fusarium moniliforme (source radix pseudostellariae).
Fig. 4 is that the cellulose degradation ability (A) of Korea S pseudomonad (Pseudomonaskoreensis) CJ-361 is conciliatePhosphorus ability (B) is evaluated. A:CK represents negative control, is Pseudomonas fluorescens, does not have cellulose degradation ability.
Fig. 5 is the optimization of Korea S pseudomonad (Pseudomonaskoreensis) CJ-361 optimum medium.
Fig. 6 is that Korea S pseudomonad (Pseudomonaskoreensis) CJ-361 prevention and control Fusarium oxysporum is infected crown princeGinseng group training seedling .Fox: only connect Fusarium oxysporum; 361-Fox: simultaneously connect Antagonistic Fungi CJ-361 and Fusarium oxysporum; Gm: only connect stringPearl sickle-like bacteria; 361-Gm: simultaneously connect Antagonistic Fungi CJ-361 and fusarium moniliforme.
Detailed description of the invention
Below in conjunction with specific embodiment, technical scheme of the present invention is described in detail. Following examples only forDescription and interpretation the present invention, and do not form the restriction to technical solution of the present invention.
Screening and the qualification thereof of embodiment 1 Antagonistic Fungi Korea S pseudomonad (Pseudomonaskoreensis) CJ-361
1, the screening of Antagonistic Fungi Korea S pseudomonad (Pseudomonaskoreensis) CJ-361
Antagonistic Fungi Korea S pseudomonad of the present invention (Pseudomonaskoreensis) CJ-361 is from continuous cropping radix pseudostellariae rootIn the soil of border, screening obtains.
1.1 pseudomonad culture medium preparations
Pseudomonad selective medium (pseudomonasselectiveisolationagar, PSIA) compound method asUnder: take 20g soybean-casein digest agar medium (soybeancaseindigestagar, SCD) (BD,USA), add 495.5mL distilled water and add thermal agitation, after fully dissolving, then add 1mL0.1%(wt/vol) crystal violet storing solution,121 ° of C autoclave sterilization 15min while being cooled to about 50 ° of C, add 3.5mL5%(wt/vol in culture medium again) furanMutter and chew pyridine storing solution, after fully mixing, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, makes pseudomonad selective medium flat board after cooled and solidified rapidly.
Wherein, 0.1%(wt/vol) compound method of crystal violet storing solution is: take 0.1g crystal violet (Sangon, onSea), be dissolved in 100mL distilled water, room temperature preservation is for subsequent use; The compound method of Nitrofurantoin storing solution is: take 5g furans appropriateBecause of (Sangon, Shanghai), join 90mLN, in dinethylformamide (Sangon, Shanghai), fully dissolve, then be settled to100mL, room temperature keeps in Dark Place for subsequent use.
1.2 pseudomonads are cultivated
Take 10g radix pseudostellariae rhizosphere soil, be dissolved in 90mL sterilized water, fully vibration shakes up, and obtains 10-1Dilution. Get 10mL10-1Dilution Soil Slurry is to the cooling sterilized water of another bottle of 90mL, and fully vibration shakes up, and obtains 10-2DilutionLiquid. Dilute successively 10-3Dilution, the soil dilution of absorption 60 μ L, is applied on pseudomonad culture medium and is placed on 30 DEG CIn constant incubator, lucifuge is cultivated 30h. Select clear and legible on culture medium and the uniform single bacterium colony of growing, be forwarded to new vacation30h left and right is cultivated in 30 DEG C of continuation of monad culture medium, is placed in 4 DEG C of of short duration saving backup of refrigerator.
1.3 Antagonistic Fungi screenings
Potato glucose culture medium (PDA), is formulated as follows: potato 200g, break into after mashed potatoes with beater, and add appropriateTiming 25min after water boil crosses four layers of filtered through gauze after it is cooled to non-scald on hand. After being collected, filtrate adds glucose30g, agar 15g, heating stirs and evenly mixs and adds water and is settled to packing after 1000mL. After 115 DEG C of autoclaving 15min, preserveFor subsequent use.
When use, the culture medium that autoclave sterilization is crossed is reheated to dissolving, treat that culture medium temperature drops to 55 DEG C of left sidesWhen right (non-scald on hand), be down flat fast plate. After culture dish fully solidifies at its bottom frame of crossing, apart from the about 2.5cm in the center of circleThe bacterium of the activated cultivation of place's inoculation, is placed in 28 DEG C of constant incubator lucifuges and cultivates after 48h, in the center of culture dishInoculation radix pseudostellariae specialized form fusarium moniliforme, is placed in 28 DEG C of constant incubator lucifuge face-offs and cultivates a couple of days, and Real Time Observation is antibacterialThe size that has that it's too late of circle. Face-off was cultivated after 4 days, filtered out a strain to radix pseudostellariae specialized form beading sickle by observing inhibition zone sizeCutter bacterium and Fusarium oxysporum have the bacterium (as shown in Figure 3) of strong antagonistic effect, and this bacterial strain has been carried out to preservation, and deposit number isCGMCCNO.12122. The strong antagonistic strain screening is carried out to purifying cultivation, and inclined-plane saves backup.
2, the qualification of Antagonistic Fungi
2.1 Antagonistic Fungis of the present invention (deposit number is: CGMCCNO.12122) can see that through microexamination this bacterium isCorynebacterium, Gram-negative bacteria. On LB solid medium, be protuberance, wavy, moistening bacterium colony. Meanwhile, the life of this Antagonistic FungiPhysiological-biochemical characteristic is: energy decomposing sucrose, fructose, sweet mellow wine, glycerine, ethanol, glucose, arginine, alanine and inositol,But do not reduce lactose, wood sugar and citric acid. Can decomposing ammonium chloride, ammonium sulfate, potassium nitrate, ammonium nitrate and urea, but can notDecompose glutamic acid. Energy hydrolyzed starch, can liquefy gelatin. The pH scope of normal growth is 5～9.5. Having stronger cellulose fallsSolution ability and phosphorus decomposing ability (as shown in Figure 4).
2.2 Molecular Identification
The strong antagonistic strain filtering out (deposit number is: CGMCCNO.12122) is forwarded to 5mlLB fluid nutrient medium to be enteredRow expands cultivates rear (28 DEG C, 180rpm) extraction DNA. Pcr amplification 16srRNA gene, identifies for bacteria molecule. Bacterium baseAs follows because organizing DNA extracting method: to get 1mL and shake the pseudomonad bacterium liquid (28 ° of C, 180rpm) spending the night, 10000rpm centrifugal 5Min, removes supernatant, adds 950 μ LTE buffer solutions suspension precipitations, and add 50 μ L10%SDS solution and 5 μ in precipitation thallineL Proteinase K (20mg/mL), mixes, 37 ° of C water-bath 1h, then add 150 μ L5mol/mLNaCl solution and 150 μ LCTAB/NaCl solution (10%CTAB, 4.1%NaCl), mixes, and 65 ° of C water-bath 20min add isopyknic Fen ︰ Lv Fang ︰ isoamylAlcohol (volume ratio 25 ︰ 24 ︰ 1) solution carries out extracting, and the centrifugal 10min of 12000rpm, by isopyknic chloroform/isoamyl for supernatantAlcohol (24/1) again extracting once, equal-volume isopropyl alcohol precipitation at room temperature 30min for supernatant, the centrifugal 10min of 12000rpm, abandonsSupernatant, DNA precipitation is cleaned with 70% ethanol, after natural air drying, dissolves with 50 μ L sterilized waters.
Adopt 16srRNA gene PCR amplification technique to carry out Molecular Identification to the Antagonistic Fungi of separation screening. Qualification primer orderClassify as: 27f(5'-AGAGTTTGATCCTGGCTCAG-3') and 1522r(5'-AAGGAGGTGATCCAGCCGCA-3'). PCR expandsIncreasing system (50 μ L) is: 25 μ LTaqPCRMasterMix (2 ×) (TransGen, Beijing), 1.0 μ L upstream and downstream are drawnThing (10 μ M), 1 μ LDNA template (20ng), 22 μ L sterilized waters. Pcr amplification program is: 94 DEG C of denaturation 5min, 94 DEG CSex change 1min, 56 DEG C of annealing 1min, 72 DEG C are extended 90sec, and 35 circulations continue 72 DEG C and extend 10min. The base of amplificationBecause fragment detects with 1% agarose gel electrophoresis, and with UniversalDNAPurificationKit glue reclaim kit(TIANGEN, Beijing) purifying reclaims object band, and serves Hai Shenggong order-checking portion and check order. Sequencing sequence adopts BLAST workTool and ncbi database (Nucleotidecollection(nr/nt)) compare, sequence is as SEQIDNO.3 instituteShow. This Antagonistic Fungi of molecular biology identification is Korea S pseudomonad (PseudomonaskoreensisCJ-361), and preservation is compiledNumber be: CGMCCNO.12122.
The fungistatic effect of embodiment 2 Antagonistic Fungis detects
Use PDA culture medium, at the Korea S pseudomonad CJ-361 apart from the activated cultivation of 2.5cm place, center of circle inoculation, be placed in 28 DEG CIn constant incubator, lucifuge is cultivated after 48h, at the center of culture dish inoculation disease fungus (spore as sharp in radix pseudostellariae specialized formSickle-like bacteria, fusarium moniliforme), be placed in 28 ° of C constant incubator lucifuge face-offs and cultivate a couple of days, the formation of Real Time Observation inhibition zone.Cultivate discovery in 5 days, Korea S pseudomonad (PseudomonaskoreensisCJ-361) efficiently antagonism radix pseudostellariae specially changesThe mycelial growth (as shown in Figure 3) of type Fusarium oxysporum and fusarium moniliforme.
Embodiment 3 Korea S pseudomonad (PseudomonaskoreensisCJ-361) optimum medium optimizations
Preparing following culture medium cultivates the most suitable growth of Korea S pseudomonad (PseudomonaskoreensisCJ-361)Base is optimized, and culture medium is as follows: 1) full LB solution; 2) 1/4LB solution; 3) 1/4LB+1/20MS; 4) 1/4LB+1/40MS;5) 1/4LB+1/80MS; 6) 1/4LB+1/20MS+0.1wt.% brown sugar; 7) 1/4LB+1/40MS+0.1wt.% brown sugar; 8) 1/4LB+ 1/80MS+0.1wt.% brown sugar.
Wherein, MS culture medium (all not containing molysite) formula is: 1) a great number of elements: 1900mg/LKNO3、1650mg/LNH4NO3、370mg/LMgSO4·7H2O、170mg/LKH2PO4、440mg/LCaCl2·2H2O; 2) trace element: 22.3mg/LMnSO4·4H2O、8.6mg/LZnSO4·7H2O、6.2mg/LH3BO3、0.83mg/LKI、0.25mg/LNa2MoO4·7H2O、0.025mg/LCuSO4·5H2O、0.025mg/LCoCl、2.6mg/LH2O; 3) organic matter: 2.0Mg/L glycine, 0.5mg/L puridoxine hydrochloride, 0.1mg/L Tyiamine Hd element, 0.5mg/L nicotinic acid, 100mg/L fleshAcid; LB culture medium prescription is: tryptone 10g/L, yeast extract 5g/L, NaCl10g/L.
Concrete operation step is as follows: the above each culture medium of 5mL accesses respectively the Antagonistic Fungi bacterium that equivalent (10 μ L) has activatedLiquid, is placed in concussion on 30 DEG C, 180rpm shaking table and cultivates 10h, takes out afterwards culture test tube, after bacterium liquid fully shakes up in 600nmRapid test light absorption value (0D under wavelength600). Result shows: the cultivation of " 1/4LB+1/20MS+0.1wt.% brown sugar " this formulaBase is the growth (as shown in Figure 5) of applicable this Antagonistic Fungi.
Embodiment 4 Antagonistic Fungi prevention and control sickle-like bacteria infringement radix pseudostellariae group training seedling effect assessments
Preparation radix pseudostellariae group training seedling MS culture medium (MS+0.5mg/L6-BA+0.3mg/LNAA+30g/L sucrose+ 7g/L agar+8g/L farina, pH=6.5), each tissue culture bottle adds 35mL culture medium, through HTHPSterilizing, after to be cooled solidifying, burns a ditch in media surface with tweezers, in ditch one side joint kind radix pseudostellariae seedling 2 strains,Be placed in 25 DEG C of constant temperature group training chambers and cultivate after 40 days, in ditch, add with optimum medium (1/4LB+1/20MS+0.1wt.%Brown sugar) the Antagonistic Fungi bacterium liquid 300 μ L of overnight incubation, control group adds equivalent LB culture medium and replaces, in the opposite side inoculation of ditchFusarium oxysporum or fusarium moniliforme, continue in ditch, to add Korea S pseudomonad CJ-361 bacterium liquid during this time, and observation is infectedSituation. Result shows, between pathogen and group training seedling, is added with in the test group of Antagonistic Fungi, Fusarium oxysporum or fusarium moniliformeGrow all suppressed, only outside ditch, in scope, grow, and add in the control group of equivalent LB culture medium, Fusarium oxysporum orThe growth of fusarium moniliforme is quick, can cross ditch, infects radix pseudostellariae group training seedling plant, causes group training seedling stem rot rotten, bladeJaundice is withered, final dead (as shown in Figure 6).
As fully visible, the strain Antagonistic Fungi (preserving number is: CGMCCNO.12122) that the present invention filters out is to radix pseudostellariae diseaseFormer bacterium has very strong antagonism, effectively prevention and control Fusarium oxysporum and fusarium moniliforme pathogen invading radix pseudostellariaeEvil, has broad application prospects in this field.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change withModify, all should belong to covering scope of the present invention.
SEQUENCELISTING
<110>University Of Agriculture and Forestry In Fujian
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ctctggaaagttcattggatgtcaaggcctggtaaggttcttcgcgttgcttcgaattaa540
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ccgtactccccaggcggtcaacttaatgcgttagctgcgccactaagagctcaaggctcc660
caacggctagttgacatcgtttacggcgtggactaccagggtatctaatcctgtttgctc720
cccacgctttcgcacctcagtgtcagtatcagtccaggtggtcgccttcgccactggtgt780
tccttcctatatctacgcatttcaccgctacacaggaaattccaccaccctctaccatac840
tctagctcgccagttttggatgcagttcccaggttgagcccggggatttcacatccaact900
taacgaaccacctacgcgcgctttacgcccagtaattccgattaacgcttgcaccctctg960
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cacccgtccgccgctgaatccaggagcaagctcccttcatccgctcgactgca1433
Claims (4)
1. a strain Korea S pseudomonad, is characterized in that: described bacterial strain is Korea S pseudomonad (PseudomonasKoreensis) CJ-361, has been preserved in the common micro-life of China Committee for Culture Collection of Microorganisms on January 28th, 2016Thing center, culture presevation is numbered CGMCCNO.12122.
2. a growth medium for Korea S pseudomonad as claimed in claim 1, is characterized in that: its growth medium is 1/4LB culture medium+1/20 is not containing molysite MS culture medium+0.1wt.% brown sugar.
3. the growth medium of a strain Korea S pseudomonad according to claim 2, is characterized in that: described not containing molysiteIts formula of MS culture medium is: 1) a great number of elements: 1900mg/LKNO3、1650mg/LNH4NO3、370mg/LMgSO4·7H2O、170mg/LKH2PO4、440mg/LCaCl2·2H2O; 2) trace element: 22.3mg/LMnSO4·4H2O、8.6mg/LZnSO4·7H2O、6.2mg/LH3BO3、0.83mg/LKI、0.25mg/LNa2MoO4·7H2O、0.025mg/LCuSO4·5H2O、0.025mg/LCoCl、2.6mg/LH2O; 3) organic matter: 2.0mg/L glycine, 0.5mg/L hydrochloric acidPyridoxol, 0.1mg/L Tyiamine Hd element, 0.5mg/L nicotinic acid, 100mg/L creatine; Described LB culture medium is: tryptosePeptone 10g/L, yeast extract 5g/L, NaCl10g/L.
4. a Korea S as claimed in claim 1 pseudomonad is replanted disease and soil-borne disease at control medicinal plant radix pseudostellariaeOn application.
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| CN109517770B (en) * | 2019-01-25 | 2021-12-21 | 黑龙江大学 | Aerobic facultative autotrophic denitrifying bacterium and application thereof |
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